Genetic relationships among salamanders of the genus Hynobius (Amphibia, Caudata) from Korea and southwestern Japan

We performed allozyme analysis for three Korean (Hynobius leechii, H. quelpaertensis, and H. yangi) and three Japanese (H. nebulosus, H. tsuensis, and H. dunni) salamanders to clarify their interspecific relationships using H. naevius as an outgroup. The genetic distances (Nei's D) within ingroup species ranged from 0.11 to 0.78 with a mean of 0.33. In the NJ and CONTML trees, monophyly of the ingroup was not supported and Korean H. quelpaertensis and H. leechii diverged first from the remaining species, which together formed a weakly supported clade. Korean H. yangi, long identified as H. leechii, was closer to Japanese H. nebulosus (D=0.108) and H. tsuensis (D=0.138) than to Korean H. leechii (D=0.197) and H. quelpaertensis (D=0.305). Hynobius tsuensis and H. nebulosus were very close (D=0.108) despite their different breeding habits. A geohistorical hypothesis is proposed to explain the divergence of the six species.     

Allozymic variation of Hynobius kimurae Dunn (Amphibia, Caudata)

An electrophoretic survey was conducted to examine genetic divergence among 21 populations of a lotic-breeding salamander Hynobius kimurae from Honshu, Japan. Genetically H. kimurae proved to be specifically distinct from H. naevius. Hynobius kimurae is divided genetically into two groups of local populations, and the five populations from the eastern area are genetically distinct from the remaining populations of the central and western areas. Less prominent genetic differentiation was revealed between the western and central populations. Separation of the eastern and central-western groups are discussed in relation to the formation of the Japanese mainland.     

Karyotype of a Japanese salamander Hynobius katoi and its implication on breeding ecology (Amphibia: Caudata)

Karyotype of a Japanese small salamander, Hynobius katoi, was first described. All individuals examined had 2n=58 chromosomes, consisting of nine pairs of biarmed macrochromosomes, four pairs of biarmed medium-sized chromosomes, six pairs of biarmed microchromosomes, and 10 pairs of uniarmed microchromosomes, although distinction of the second and the third groups of chromosome pairs was not clear. All pairs appeared homologous and no sexual dimorphism was found. Possession of 2n=58 chromosomes in H. katoi strongly suggests its lotic-breeding habits as was expected from the number and size of eggs and adult morphology. When compared morphology of chromosomes among lotic-breeders with 2n=58 chromosomes, metacentric nature of No. 10 seems to characterize the karyotype of H. katoi.     

A new species of salamander of the genus Hynobius from Central Honshu, Japan (Amphibia, Urodela)

We describe a small salamander from south Central Honshu, Japan, as a new species, Hynobius katoi. The genetic distances between this species and several named species, including sympatric H. kimurae, derived from allozyme data from a starch gel electrophoresis, proved to be sufficiently large to differentiate it at a specific rank. Distribution of this species is confined to the montane regions of Shizuoka and Nagano Prefectures, on the Akaishi Mountains of the Chubu District, central Japan. It is regarded as a member of the naevius group of Hynobius, characterized by small number of large, pigmentless ova. The species differs from the other species of the naevius group by the combination of relatively small body size, nearly spotless body, relatively few vomerine teeth forming moderately shallow series, and unique electrophoretic pattern of isozymes.     

中国小鲵的核型

中国小鲵(Hynobius chinensis)被描述于116年前,我国学者于2005年在模式产地湖北宜昌再次采到标本,至此该物种的有效性得以确认。以2007年采于模式产地的中国小鲵胚胎为材料,采用Giemsa染色方法,对中国小鲵的核型进行了分析。结果表明,中国小鲵的二倍体染色体数为56,28对染色体按大小和形状可以分为4组:第一组,包括Nos.1～9,由大型m,sm,st染色体组成;第二组为中型双臂染色体组,由Nos.10～13组成,包含m,sm染色体;第三组,包括Nos.14～18,由小型m染色体组成;第四组,包括Nos.19～28,由小型t染色体组成。与小鲵属其他物种相比,中国小鲵核型与具染色体数为2n=56的类群核型大致相同,差异出现在染色体分组和相应染色体着丝点类型上。     

Taxonomic status of a salamander species allied to Hynobius naevius and a reevaluation of Hynobius naevius yatsui (Amphibia, Caudata)

A Japanese small salamander, Hynobius naevius (Temminck and Schlegel, 1838), has recently proven to include two species (larger [A] and smaller [B]) that are reproductively isolated and are genetically and morphologically distinct from each other. Of these two species, Species A is considered to correspond to true H. naevius. To determine the name of Species B, we investigated five specimens stored at Kyushu University and concluded from the locality and date of collection that they represent syntypes of H. n. yatsui Oyama, 1947. Results of morphological comparisons indicated that the syntypes are identical with Species B. Because this species is distinct from Hynobius naevius at the species level, subspecies H. n. yatsui is elevated to full species rank as H. yatsui. To avoid taxonomic confusion, we designated one male among the syntypes as the lectotype and redescribed the species.     

A skeletochronological study of breeding females in a population of Japanese clouded salamanders (Hynobius nebulosus)

The age structure of breeding females of Hynobius nebulosus has not been studied sufficiently. We estimated the ages of 76 individuals from a population in Kyoto by using skeletochronology. The mean age and snout-vent length (SVL) of this population were 4.6 years and 55.7 mm, respectively. It was estimated that the youngest females breed two years post hatching at a mean SVL of 46.5 mm, but a larger number of individuals begins breeding at three years and a mean SVL of 52.2 mm. Because most males also start to breed at three years, there seems to be no gender difference in the timing of sexual maturation. The age of the oldest female was estimated to be 11.8 years. It is possible that the life history of H. nebulosus is characterized by early maturation and arrested growth, and short longevity.     

挂榜山小鲵的核型(有尾目:小鲵科)

本文首次报道了挂榜山小鲵Hynobius guabangshanensis 的核型.其2n=56,分为4组,其中9对大型染色体,4对双臂中型染色体,5对双臂小型染色体和10对单臂小型染色体.挂榜山小鲵具有池塘类型小鲵的特征,即2n=56以及缺少中端着丝粒染色体,这与挂榜山小鲵的生态和形态特征也属池塘类型相符.     

三种小鲵和极北鲵的核型

本文报道了安吉小鲵（Ｈｙｎｏｂｉｕｓａｍｊｉｅｎｓｉｓ）、中国小鲵（Ｈ．ｃｈｉｎｅｎｓｉｓ）、东北小鲵（Ｈ．ｌｅｅｃｈｉ）（辽宁桓仁）和极北鲵（Ｓａｌａｍａｎｄｒｅｌａｋｅｙｓｅｒｌｉｎｇｉ）（吉林白河）的核型。３种小鲵２ｎ＝５６,染色体按大小和形状分为大、中、小型双臂和小型单臂４组。极北鲵２ｎ＝６２,染色体按大小和形状可分为大、中、小型３组。对东北小鲵辽宁桓仁与辽宁千山标本核型的显著差异结合分类学问题进行了讨论。对３种小鲵核型进行了比较分析,并讨论了中国的小鲵属核型特点。比较了不同地区极北鲵核型的异同,并将其与小鲵属核型进行了比较。     

Phylogenetic relationships and phylogeography of Hynobius tokyoensis (Amphibia: Caudata) using complete sequences of cytochrome b and control region genes of mitochondrial DNA

Using complete sequences of cytochrome b (cyt b) and control region (CR; D-loop) genes of mitochondrial DNA, we investigated the phylogenetic relationships and phylogeography among populations of the salamander Hynobius tokyoensis in northern Japan. Inclusion of populations from Aichi, Chubu region within Hynobius nebulosus by previous results was confirmed because they formed a clade with H. nebulosus. Monophyly of H. tokyoensis was supported in all trees analyzed, and two clades consistently emerged: clade A from northeastern Kanto (Ibaraki) to southern Tohoku (Fukushima), and clade B from the remaining area south of northern Kanto (Tochigi). These two clades are estimated to have an old history of divergence during the late Pliocene, when the present-day Kanto Plain was under the sea. Within clade B, three haplotypes groups (I-III) are hypothesized to have diverged more recently, i.e., between the early and mid-Pleistocene, probably in relation to glacial events. The ancestral groups I and II were separated at the present-day western Kanto and Boso Peninsula areas, respectively, and subsequently, group II invaded northward to the present-day northern Kanto area and gave rise to group III. Populations of the Miura Peninsula are thought to have multiple origins, one directly from Boso and another from a more recent southward invasion from the northern Kanto area. Cytochrome b appears to have evolved more quickly than CR genes in H. tokyoensis, and thus will be more useful for phylogenetic analyses and formulating conservation measures.     

A new species of lentic breeding Korean salamander of the genus Hynobius (Amphibia, Urodela)

A new species of salamander, Hynobius yangi, is described from Kori (=Hyoam-ri), Busan-shi, southeastern Korea. It is a lentic breeder belonging to the H. nebulosus species group, and has long been confused with another Korean species H. leechii. The new species, however, is genetically substantially differentiated from Korean and Japanese relatives. Hynobius yangi is morphologically very similar to H. leechii, but could be differentiated from it by the tail shape, degree of limb separation, shape of vomerine teeth series, shape of egg sac, and dorsal coloration.     

Estimation of age structure by skeletochronology of a population of Hynobius nebulosus in a breeding season (Amphibia,Urodela)

Using skeletochronology, we determined the age structure of adult Hynobius nebulosus from Kyoto in the breeding season of 1998. From previously marked individuals, the lines of arrested growth proved to be formed once per year, indicating the number of winters each salamander experienced. The age at first reproduction was estimated to be 2.8-2.9 yrs of age in males and 3.8-3.9 yrs in females. The oldest males and females were 9.8-9.9 and 5.8-5.9 yrs of age, respectively, and, therefore the longevity in this species was estimated to be more than 9 yrs for males and 5 yrs for females. The growth curve of male's body size estimated indicated that the growth rate much decreases after males attained sexual maturity. Because body sizes of adults greatly vary even within an age class, it is dangerous to estimate individual age from the size frequency data at least in adults. We discussed age properties in Hynobius by comparing lentic and lotic breeders.     

Food habit of the juvenile of the Japanese newt Cynops pyrrhogaster

The previous study showed that the red coloration of the ventral skin of the Japanese newt Cynops pyrrhogaster was associated with the number of carotenoid vesicles and the content of carotenoid in the pigment cell of the skin. To elucidate the mechanism for the red coloration of the skin of the newt, we studied the food habit of the juvenile from the Japanese newt Cynops pyrrhogaster. Sixty-two juveniles were collected in Fukue Island in Nagasaki Prefecture from November 2000 to May 2002 and divided into 2 groups according to the snout-vent length (SVL). Over 400 prey animals were obtained from the juveniles by stomach flushing. In the larger group (SVL>30.0mm), Collembola (45.4%) and Acari (12.6%), which are very common species of soil animals, were the prey animals dominant in number. In the group with the smaller SVL (<29.9mm), Collembola (30.4%) and Acari (25.4%) were in number as well. We also studied the food habit of the Japanese clouded salamander, Hynobius nebulosus. In the salamander, Doratodesmidae (56.5%) and Amphipoda (13%) were the prey animals dominant in number. Our results, taken together, suggest that the Japanese juvenile C. pyrrhogaster does not change its food habit as it grows, and that it eats soil animals common in its habitat. Moreover, the food habit of juvenile C. pyrrhogaster differs from that of H. nebulosus, although the juveniles of both species live in the same area.     

Evidence that the voltage-dependent component in the fertilization process is contributed by the sperm

To investigate the mechanisms that account for the voltage dependence of fertilization and provide an electrical block to polyspermy, we studied cross-fertilizations between three species of amphibians having different degrees of voltage dependence. Anurans, such as the toad Bufo japonicus, as well as the primitive urodele Hynobius nebulosus, have voltage-dependent fertilization; other urodeles, such as Cynops pyrrhogaster, have voltage-independent fertilization (Y. Iwao, 1989, Dev. Biol. 134, 438-445). Entry of Hynobius sperm into Cynops eggs was blocked by clamping the egg's membrane potential at +40 mV, as is the case for fertilization of Hynobius eggs with Hynobius sperm, but not for fertilization of Cynops eggs with Cynops sperm. Therefore, fertilization was voltage dependent in an experimental condition where only the sperm could be contributing this characteristic. The voltage-dependent properties of fertilization between Bufo eggs and Hynobius sperm were also characteristic of the sperm species; fertilization was blocked at +50 mV as in Hynobius fertilization, but not at +20 mV as in Bufo fertilization. These results support the conclusion that the voltage dependence of fertilization results from a component contributed by the sperm.     

Geographic allozyme variation in the Japanese clouded salamander, Hynobius nebulosus (Amphibia: Urodela)

Using starch gel electrophoresis, geographical enzyme variation was surveyed in 46 populations of the Japanese salamander Hynobius nebulosus , which occurs widely in western Japan. This species exhibits substantial local genetic differentiation and is more diverse in inland regions than on small islands. In several different analyses, two groups of populations, one from Kyushu Island to the westernmost part of Chugoku district, Honshu (western group), and another from Shikoku through Kinki to Chubu district, Honshu (eastern group), were consistently recognized. Most of the remaining populations were placed in two less clearly-defined groups: the montane group from the Chugoku Mountains and the Chugoku group from the coastal regions of Chugoku district. The divergence patterns of H. nebulosus are thought to be related to the geological history of its range. The populations near the boundary of the western group were found to share several alleles that were predominant on both sides of the boundary, indicating past secondary contact. Differentiation in the montane group did not follow the isolation by distance model, probably because several populations included were genetically mixed with lowland populations of the Chugoku group.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 89 , 311–330.     

Phylogenetic relationships of Hynobius naevius (Amphibia: Caudata) as revealed by mitochondrial 12S and 16S rRNA genes

Using mitochondrial 12S and 16S rRNA sequences, we investigated phylogenetic relationships among populations of the endemic Japanese salamander Hynobius naevius. Monophyly of this species was recovered only in the maximum parsimony tree and was unresolved in maximum likelihood and Bayesian trees. Instead the following four haplotype clades consistently emerged clearly: Clade 1 from northwestern Kyushu, Clade 2 from Chugoku and northeastern Kyushu, Clade 3 from western Shikoku and Kyushu, and Clade 4 from Chubu-Kinki and central-eastern Shikoku. Of these, Clades 1 and 2, and Clades 3 and 4, respectively, correspond to Groups A and B previously recognized from the analyses of allozyme data in this species, but monophyly of these groups was not strongly supported. Unlike the previous results, the western and eastern samples from Shikoku did not form a clade, and were grouped with Kyushu-B in Clade 3 and Chubu-Kinki in Clade 4, respectively. The reason for this conflict between mtDNA and allozyme results is unknown, but might be related to retention of ancestral mtDNA polymorphism in Shikoku populations. Nearly simultaneous divergence of as many as four lineages in wide-ranging H. naevius is inferred for the late Miocene-Pliocene history of this taxon.     

Karyotypes of two rare species of hynobiid salamanders from Taiwan,Hynobius sonani (Maki) and Hynobius formosanus Maki (Urodela)

Two salamanders endemic to Taiwan, Hynobius sonani and Hynobius formosanus, are similar to Japanese hynobiids in having 2n = 58 chromosomes and by the absence of a medium-sized telocentric pair. H. sonani has 6 metacentric and 10 telocentric microchromosome pairs, while H. formosanus has 5 metacentric and 11 telocentric microchromosome pairs. The karyotype of H. sonani is nearly identical to that of the Japanese hynobiid salamander H. boulengeri. Karyophylogenetic relationships among forms of mountain-stream type hynobiids of Taiwan and Japan are discussed.This paper is dedicated to late Mr. Tadao Yamamoto who died on November 2, 1985.     

Immunocytochemical survey of the neuropeithelial endocrine system in the respiratory tract of the Tokyo salamander,Hynobius neubulosus tokyoensis Tago

The epithelial lining of the respiratory tract of urodeles has been shown to harbor an innervated system of neuroepithelial endocrine (NEE) cells. Even between phylogenetically closely related species, large differences have been reported in the appearance and chemical coding of the NEE system. Although urodeles are well suited for the purpose, none of the prior studies have provided an immunocytochemical survey of the NEE system in all parts of the respiratory tract. In the present study, many bioactive substances and a general marker were immunocytochemically demonstrated in serial sections of the entire respiratory tract of the Tokyo salamander, Hynobius nebulosus tokyoensis, a species in which neuroepithelial bodies (NEBs) were previously characterized at the electron microscopic level. In the current study, serotonin-immunoreactive solitary NEE cells were observed in variable numbers in the larynx, in all parts of the trachea, and in areas of the lungs covered with ciliomucous epithelium. Serotonin-containing NEBs, however, were detected in small cranial areas of the lung only. Solitary NEE cells were seen in the trachea and lungs of H. nebulosus tokyoensis by immunocytochemical staining for somatostatin, calcitonin, calcitonin gene-related peptide, and bombesin, but the number, localization, and appearance of the labeled NEE cells differed considerably. Only calcitonin-like immunoreactivity was also noted in some NEB-like cell clusters in the cranial parts of the lungs. Unlike many other vertebrates, neuron specific enolase was found to be a poor marker for the NEE system in the salamander species used in this investigation. It may be concluded that the NEE system of H. nebulosus tokyoensis contains at least five different bioactive substances. The different markers, however, demonstrate the presence of NEE cells with obvious differences in respect to appearance and topographical distribution. The necessity is emphasized of reliable methods for adequate sampling of all regions of the respiratory tract in comparative histological studies of the NEE system.     

An electrically mediated block to polyspermy in the primitive urodele Hynobius nebulosus and phylogenetic comparison with other amphibians

At fertilization, the egg of the primitive urodele, Hynobius nebulosus, produced a fertilization potential which rose from -12 to +47 mV. A similar activation potential was elicited by pricking with a needle, by applying A23187, or by electric shock. The potential change was mediated by an increased permeability to Cl-. Clamping the egg's membrane potential at +40 mV blocked fertilization, while clamping at +20 mV induced polyspermy. These results indicated the occurrence of an electrical polyspermy block, typical of anurans, but atypical of urodeles. Furthermore, Hynobius eggs fertilized by natural mating incorporated only one sperm nucleus, and experimentally polyspermic eggs underwent multipolar division. Accessory sperm did not degenerate in the egg cytoplasm, indicating lack of an intracellular polyspermy block. By comparison, fertilization of Bufo japonicus (anuran) was also voltage dependent, whereas that of Cynops pyrrhogaster (urodele) was voltage independent. Thus polyspermy prevention mechanisms in Hynobius closely resemble those of anuran amphibians and differ from those of higher urodeles.